A major area of interest in genome biology is targeted integration of one or more sequences of interest into desired locations. Attempts have been made to alter genomic sequences in cultured cells by taking advantage of the natural phenomenon of homologous recombination.
If a polynucleotide has sufficient homology to the genomic region comprising the sequence to be altered, it is possible for part or all of the sequence of the polynucleotide to replace the genomic sequence by homologous recombination.
However, the frequency of homologous recombination under these circumstances is extremely low. Moreover, the frequency of insertion of the exogenous polynucleotide at genomic locations that lack sequence homology exceeds the frequency of targeted homologous recombination by several orders of magnitude.
The introduction of a double-stranded break into genomic DNA, in the region of the genome bearing homology to an exogenous polynucleotide, has been shown to stimulate homologous recombination at this site in cultured cells.
However, the question of where to introduce new genes remains problematic. For some purposes, it is commonly thought that integration should be targeted to genes that are thought to be dispensable or to extragenic regions.
Genomic safe harbours (GSHs) are intragenic or extragenic regions of the human genome that are able to accommodate the predictable expression of newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbour must permit sufficient transgene expression to yield desired levels of the vector-encoded protein or non-coding RNA. A GSH also must not predispose cells to malignant transformation nor alter cellular functions.
Only three sites in the human genome have been used for targeted transgene addition to date: the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCRS) gene locus, and the human orthologue of the mouse ROSA26 locus. At present, the information that is currently available regarding the safety features of these loci is too limited to qualify any of them as a GSH.
However, there remain needs for compositions and methods for stable targeted integration into a locus within the genome that provides constitutive expression.